Ex vivo electrophysiology

Deeply anesthetize the mouse with isofluorane, decapitate, and remove the brain.

Using a vibratome, cut 150mm horizontal slices containing the region of interest in ice-cold aCSF solution and allow to recover at 33˚ C in aCSF for at least one hour.

For fluorescent imaging, visualize slices under an Axio Examiner A1 equipped with Dodt and IR optics using a Zeiss Axiocam 506 mono and Neruolucida 2023 software.

Whole-cell patch-clamp recordings are made at 33˚ C using 3 – 5 MOhm pipettes. Recordings are made using Sutter IPA and SutterPatch v2.3.1 software (Sutter Instruments), filtered at 5 kHz and collected at 10 kHz.

For I _h: voltage clamp cells at -60mV and step to -40, -50, -70, -80, -90, -100, -110, and -120 mV. I _hmagnitude is quantified as the difference between the initial steady-state response to the -120mV step and the asymptote of the slow current sag.

For spontaneous firing rates: record in current-clamp mode ( I = 0 pA). Spontaneous firing rate is measured as the mean firing rate during the first 2 min of whole-cell recording.

Action potential (AP) waveform measurements are made from averages across at least 8 APs from the first 2 min of recording.

For input resistance: Apply a brief hyperpolarizing pulse once every 10 sec and average across the measurements made during the first 2 min of recording.

For CNO testing in DREADD-expressing neurons: spontaneous firing rate or resting membrane potential are monitored until a stable baseline is observed for at least 5 min. Then switch the perfusion solution to 1mM CNO for 5 min.

When recordings are complete, drop fix slices in 4% formaldehyde in PBS for at least 2 hr.

Complete statistical analyses in R, first testing whether data meet the criteria for parametric statistical evaluation.